Tobacco and tobacco packaging material for preventing or reducing tobacco-associated injury in the aerodigestive tract of a subject

ABSTRACT

Articles of manufacturing for preventing or reducing tobacco smoke- and smokeless-tobacco associated injury in the aerodigestive tract of a subject are disclosed, which comprise at least one agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract, said at least one agent being selected from the group consisting of a cyanide chelator and an iron chelator.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of preventing or reducingpathogenesis of oxidant stress-associated diseases of the aerodigestivetract. More particularly, the present invention relates to methods ofemploying hydroxocobalamin (vitamin B12a, OH—CO), deferoxamine (DES) andreduced glutathione (GSH) to reduce or prevent tobacco-induced cellularor macromolecular damage in the aerodigestive tract.

Harmful Effects of Tobacco Consumption:

The deleterious effects of tobacco abuse are well known. Tobacco is aworldwide public health hazard accounting for significant morbidity andmortality. Although smoking places an abundant oxidant insult to theoropharynx and respiratory tract, the oxidant burden associated with anytobacco consumption (as described hereinbelow) is deleterious to theentire body of the tobacco consumer. Namely, tobacco consumption leadsto development or enhancement of atherosclerosis, cardiovasculardisease, chronic obstructive pulmonary disease and various forms ofcancer, including carcinomas of the mouth, pharynx, esophagus and lung.

There are two principal ways to consume tobacco: smoking and smoke-lessconsumption. The latter includes spit tobacco, chewing tobacco, chew,chaw, dip, plug, comes in various forms: snuff, snuss and chewingtobacco. Snuff is a fine-grain tobacco that often comes in teabag-likepouches, which users “pinch” or “dip” between their lower lip and gum.Chewing tobacco comes in shredded, twisted, or “bricked” tobacco leavesthat users put between their cheek and gum. Whether ifs snuff, snus orchewing tobacco, the user consumes the tobacco letting it sit in themouth and suck on the tobacco juices, spitting often to get rid of thesaliva that builds up. This sucking and chewing allows nicotine (anarcotic drug), to be absorbed into the bloodstream through the tissuesof the mouth. Smokeless tobacco has a detrimental effect on the oralcavity plus systemic effects from buccal absorption of nicotine andother chemicals.

Approximately 50 million Americans smoke and countless others areaffected by tobacco smoke (TS) as secondary smokers. Children of smokersbreathe this second-hand smoke and have more respiratory problems thanchildren of non-smokers.

Studies have estimated that TS has over 3,000 different constituents, ofwhich many are toxic, carcinogenic and/or generate free radical species.Free radicals are atoms or molecules containing an unpaired electron.Oxygen free radicals include the superoxide free radical (.O2⁻) and thehydroxyl radical (OH.) which, together with hydrogen peroxide (H₂O₂) andsinglet oxygen (¹O2), are jointly called reactive oxygen species (ROS).Due to their high reactivity they may lead to chemical modification andimpairment of the components of living cells, such as proteins, lipids,carbohydrates and nucleotides.

Tobacco smoke mediated oxidant injury is similar to that induced bysmog, thereby increasing such noxious stimuli to primary and secondarysmokers in polluted atmospheric environments.

Most of constituents of TS have been identified in so-called mainstreamand side stream TS. The former is that volume of smoke drawn through themouthpiece of the tobacco product during puffing while side stream smokeis that smoke emitted from the smoldering cigarette in between puffs.Although tar and nicotine are retained in the filter of cigarettes, thisapplies mainly to mainstream smoke, when comparing filter and non-filtercigarettes. Mainstream smoke emission is also markedly reduced both inlow and in ultra low tar yield cigarettes. However, the emissions oftoxic and carcinogenic components in side stream smoke are notsignificantly reduced in filter cigarettes when compared to non-filtercounterparts. Thus, side stream smoke is a major contributor toenvironmental smoke, affecting both the smoker and their non-smokingcounterparts, so called secondary smokers.

Evidence shows that cigars as well as cigarettes are highly toxic andaddictive. Tobacco smokers have a similar increased risk for oral andlaryngeal cancers. Evidence indicates that one cigar generates levels ofcarcinogenic particles exceeding those generated by three cigarettes.Fumes from cigars are also of greater consequence to secondary smokers.Epidemiologic studies reveal greater frequencies of heart disease,emphysema, and cancers of the mouth and pharynx in cigar smokers whencompared to matched non-smokers. Cigar smokers may spend one full hoursmoking a single large cigar and commonly hold an unlit cigar in themouth, allowing further exposure to toxins by local absorption. Thus,consumption of cigars may produce an equal or greater burden of toxicexposure to TS than cigarettes. Recently, sales of cigars have risen,partly due to their gaining popularity with women and the advent of thefemale friendly “cigar bar”.

Oral diseases associated with tobacco consumption: Tobacco, whethersmoked or chewed, causes common untoward effects in the oral cavity.Tobacco smoke has two chances to exert its deleterious effects in themouth; when it is inhaled by the smoker and on its exit duringexhalation.

Over 30,000 new cases of cancer of the oral cavity are diagnosedannually, accounting for 2-4 percent of all new cancers. Oral cancerkills 8,000 patients each year and only half of cases diagnosed annuallyhave a five-year survival. The great majority of these patients areusers of tobacco products. Oral squamous cell carcinoma (SCC) is themost common malignancy of the head and neck with a worldwide incidenceof over 300,000 new cases annually. The disease is characterized by ahigh rate of morbidity and mortality (approximately 50%) and in thisrespect is similar to malignant melanoma (1-4). The major inducer oforal SCC is exposure to tobacco which is considered to be responsiblefor 50-90% of cases world-wide (5, 6). As such, the incidence of oralSCC in tobacco smokers is 4-7 times higher than in non-smokers (7, 8).Moreover, the higher TS-related risk for oral SCC is manifested by areduction in the mean age of development of the disease by 15 years ascompared to non-smokers (9). Various malignancies are particularlyassociated with smokeless tobacco consumption. These include oral cancerand cancer of the gastrointestinal tract including esophagus andbladder. Leukoplakia, a tobacco induced white patch on the buccalmucosa, as found in smokers, is a localized irritation due to directcontact of smoked or smokeless tobacco and it is directly related to thefrequency and years of tobacco abuse. Although leukoplakia is a benignoral lesion, it has a malignant potential.

In addition, tobacco contributes to other oral symptoms or pathologiesof the mouth and teeth. Tobacco may cause halitosis, may numb the tastebuds, and interfere with the smell and the taste of food. It may stainteeth and contribute to dental caries. Smokers have more dental tartar(calculus) than non-smokers. Tobacco is associated also with destructiveperiodontal (gum) disease and tooth loss. Acute necrotizing ulcerativegingivitis (“trench mouth”) is a destructive, painful inflammatorycondition occurring mainly in tobacco smokers. Swelling of the nasal andsinus membranes have also been associated, purportedly, in individualswho are “allergic” to TS.

Oral submucous fibrosis occurs mainly in India and is a chronic,progressive premalignant condition. The etiology is chronic chewing oftobacco or areca nut or both. The fibrosis results in restriction ofmouth opening and involves the palates, tonsillar fossa, buccal mucosaand underlying muscle. Associated with this condition is alsooropharyngeal carcinomas, also with a high frequency in India andassociated in 70% of cases with chewing tobacco. Smokeless tobacco andareca nut usage is also common in Pakistan, Bangladesh and Java and inthese and Indian immigrants to the United States and United Kingdom.

Tobacco smoke also affects the skin adversely. Dr. Douglas Model ofEngland in 1985 added to the medical lexicon the term “smoker's face”from a study with pictures of 116 cases and suitable non-smokingcontrols (10). Akin to photodamage, those with smoker's face appearolder and have more wrinkles.

Molecular damage resulting from exposure to TS: Tobacco smoke inducesoxidative damage to lipids, DNA and proteins, particularly viaprotein-SH groups as a consequence of containing high levels of bothfree radicals as well as aldehydes, including acetaldehyde (ethanol),propanol and acrolein, as well as other deleterious molecules.

Oxidant injury: Tobacco smoke is divided into two phases; tar andgas-phase smoke. Tar contains high concentrations of free radicals. Manytar extracts and oxidants are water-soluble and reduce oxygen tosuperoxide radical which can dismutate to form the potent oxidant H₂O₂.Oxidants in gas-phase smoke are reactive carbon- and oxygen-centeredradicals with extremely short half lives.

Cells subjected to oxidative stress develop severely affected cellularfunction and suffer damage to membrane lipids, to proteins, tocytoskeletal structures and to DNA. Free radical damage to DNA has beenmeasured as formation of single-strand breaks, double-strand breaks andchromosomal aberrations. Cells exposed to ionizing radiation and TS havealso been demonstrated to have an increased intracellular DNA damage, aprecursor of mutations and development of malignancies. It has beenshown that TS elicits protein carbonylation in plasma and that, incontrast, exposure of human plasma to gas-phase but not to whole TSproduces oxidative damage to lipids.

Redox-active metals: Redox-active metal ions, such as iron and copper,in the presence of H₂O₂ and other low-reactive free radicals found inTS, such as superoxide radicals, participate in the deleteriousHaber-Weiss and Fenton reactions, in which the highly reactive hydroxylfree radicals are produced.

Aldehydes: Studies have shown that exposure of plasma to TS results inprotein damage in the form of protein carbonylation (11) and inoxidation of plasma lipids and antioxidants (12). The source of theaccumulation of protein carbonylation was found to be due to aldehydespresent in TS (13, 14). In addition, it was shown that several salivaryenzymes such as amylase, lactic dehydrogenase (LDH), and acidphosphatase were considerably affected by TS (14, 15), where bothTS-based aldehydes, such as acrolein and crotonaldehyde, as well asoxygen free radicals were implicated as the causative agents affectingthe above enzymes (14, 15).

Physiological Antioxidants:

Glutathione: Glutathione, a sulfur-containing tripeptide(L-γ-glutamyl-1-cysteine-glycine) is the most abundant non-protein thiolin mammalian cells and is recognized as the primordial antioxidant.Glutathione, in its reduced form, “GSH”, acts as a substrate forglutathione-S-transferase and glutathione peroxidase, enzymes catalyzingreactions involved in detoxification of xenobiotic compounds and inantioxidation of ROS and other free radicals. This ubiquitous proteinplays a vital function in maintaining the integrity of free radicalsensitive cellular components. Under states of GSH depletion, includingmalnutrition and severe oxidative stress, cells may then become injuredfrom excess free radical damage and die.

Oral peroxidase: Oral peroxidation is the pivotal enzymatic activity ofthe salivary antioxidant system (16-19). Oral peroxidase activity iscomposed of the combined activity of two peroxidases, salivaryperoxidase (SPO) and myeloperoxidase (MPO). Salivary peroxidase, whichis secreted by the major salivary glands, mainly the parotid gland (18),contributes 80% of the total oral peroxidase (OPO) activity, while MPO,produced by leukocytes (20), contributes the remaining 20% of OPOactivity. Oral peroxidase performs two functions preventing oxidantinjury; it reduces the level of H₂O₂ excreted into the oral cavity fromthe salivary glands, by bacteria and by leukocytes, and it inhibits themetabolism and proliferation of various bacteria in the oral cavity.

Oral peroxidase is involved in destroying TS-associated H₂O₂. Tobaccosmoke-associated hydrocyanic acid (HCN) is metabolized by the liver tothiocyanate ion (SCN⁻). This SCN⁻ is specifically sequestered from theplasma by the parotid gland and is secreted by this gland into the oralcavity. Its concentration in the saliva of non-smokers ranges from0.3-1.5 mM, while the respective range in smokers is approximately1.4-4.0 mM, depending on the number of cigarettes smoked per day, with aprolonged t_(1/2) of 9.5 h (21). Following its secretion in saliva, SCN⁻reacts with, and eliminates H₂O₂ in the oral cavity in a reactioncatalyzed by OPO, as described in FIG. 2 a. However, it has been shownthat if OPO is damaged or depleted, as occurs upon exposure to TS, theH₂O₂ in the oral cavity is not eliminated and remains available forfurther reaction with redox-active metal ions which are secreted via theparotid gland saliva (22, 23).

In the reaction of SCN⁻+H₂O₂→OSCN⁻+H₂O, which is catalyzed by OPO, H₂O₂oxidizes SCN⁻, a detoxification product of cyanide secreted mainly bythe parotid gland. In this reaction, SCN⁻ acts as the electron-donatingcomponent, similarly to GSH in other biological systems (20, 24, 25).Two potent antibacterial oxidizing products evolve from this reaction:hydrogen hypothiocyanite (HOSCN) and its conjugated anion, OSCN⁻. Theantibacterial activity of HOSCN and OSCN⁻ stems from their ability toreact with sulfhydryl groups of bacterial enzymes that are vital forglycolysis, such as hexokinase, aldolase and pyruvate kinase (20,25-28).

The importance of OPO in oral disease prevention has been demonstratedin several studies. For example, studies using animal models or the Amestest have shown that saliva inhibits the mutagenicity of known oralcancer inducers, such as TS, 4NQO and benzopyrene (29, 30). Biochemicalstudies have also demonstrated that saliva inhibits production of ROSsuch as superoxide free radical and H₂O₂ from betel quid tobacco, themost potent inducer of oral cancer (31). These observations are furthersupported in the observation that patients with oral lichen planus, apremalignant lesion, have reduced salivary antioxidant capacity (32).

Several prior art approaches have been employed in order to reduce orprevent incidence of oral disease resulting from oxidant injury.

For example, cigarette filters are used to trap TS tar but do not thegas-phase compounds.

One approach has employed a filter for TS providing chemo-sorptiveproperties to reduce aldehyde concentration in TS (33).

Another approach has employed oral megadoses of antioxidants in attemptsto reduce generation of H₂O₂ resulting from the “respiratory burst”reaction associated with phagocytic activity of macrophages andneutrophils. It has been shown that smokers have a higher “respiratoryburst” reaction than non-smokers and that this may be associated withthe increased incidence of aerodigestive tract disease in the former.

In yet another approach, dipeptide compounds with pharmaceuticalproperties to increase glutathione levels were employed (34).

A further approach utilized a glycine carboxylic acid alkyl mono-esterof glutathione to increase cellular GSH levels (35).

In yet a further approach, administration of a combination ofglutathione and selenium was suggested for preventing oxidant injuryresulting from exposure to TS (36).

In another approach, administration of a combination of glutathione,ascorbic acid, selenium and a sulfur-containing amino acid was suggestedin order to prevent oral oxidant injury (37).

In yet another approach, administration of a combination including someor all of the following antioxidants; L-glutathione, L-selenomethionine,L-selenocysteine, ascorbyl palmitate, ascorbic acid esters, L-cysteine,N-acetyl-1-cysteine, tocopherol acetate, tocopherol succinate, vitaminA, a zinc salt, methionine and taurine was suggested in order to provideintra-oral protection from oxidant injury (38)

The present inventors have previously described novel smoking filtersand oral compositions for reducing tobacco associated damage in theaeurodigestive tract (see U.S. Pat. No. 6,789,546). These compositionsinclude active agents which are capable of reducing or preventingtobacco associated loss of peroxidase activity in the aerodigestivetract.

U.S. Pat. No. 5,922,346 teaches a composition for reducing free radicaldamage induced by tobacco products and environmental pollutantscomprising, as active ingredients, reduced glutathione and a source ofselenium selected from the group consisting of elemental selenium,selenomethionine and selenocysteine, the active ingredients beingcombined with suitable carriers and flavorings for their intra-oraladministration as gels, lozenges, tablets and gums in concentrations forreducing free radical damage induced by tobacco products and otherenvironmental pollutants to the oral cavity, pharynx and upperrespiratory tract of a user and secondary smokers.

U.S. Pat. No. 5,906,811 teaches a method for reducing free radicaldamage induced by tobacco products and environmental pollutantscomprising administering in a suitable carrier in concentrations foreffectively reducing said free radical damage to the oro-pharynx andupper respiratory tract of a user a combination of from 0.01 and 10%(weight) glutathione, from 1.0 to 25% (weight) ascorbic acid, from 0.001to 10% (weight) of a source of selenium and from 0.001 to 2.0% (weight)of a sulphur containing amino acid.

These aforementioned attempts to reduce tobacco damage are used as anadjuvant treatment following or prior to tobacco consumption, but notconcomitantly with tobacco consumption.

U.S. Pat. No. 5,829,449 teach a composition for inclusion within acigarette, cigar or pipe tobacco for reducing free radical damage to theoro-pharyngeal cavity, respiratory tract and lungs from tobacco smoke,said composition comprising L-glutathione and a source of seleniumselected from the group consisting of L-selenomethionine andL-selenocysteine. U.S. Pat. No. 5,829,449 clearly states that thecomposition is supplied by smoke inhalation and not by direct contactwith the aerodigestive tract (i.e., wet tissue).

U.S. Pat. No. 6,138,683 teaches a composition for inclusion within aquantity of smokeless tobacco, selected from the group consisting ofchewing tobacco and snuff, for reducing free radical induced damage tothe oro-pharyngeal cavity of the user, said composition comprisingL-glutathione and a source of selenium in combination with saidsmokeless tobacco.

SUMMARY OF THE INVENTION

According to the present invention there is provided a smoking productcomprising an agent being capable of reducing or preventing tobaccoassociated death of cells in the aerodigestive tract of a subject, thesmoking product being designed and configured so as to enablephysico-chemical interaction between said agent and said tobacco smokewhen in use by the subject.

According to one aspect of the present invention there is provided, anarticle of manufacturing comprising tobacco and a tobacco packagingmaterial, at least a portion of said tobacco and/or tobacco packagingmaterial being in contact with an aerodigestive tract of a subject inuse thereof, and whereas said at least a portion of said tobacco and/ortobacco packaging material comprises at least one agent capable ofreducing or preventing tobacco associated loss of peroxidase activity insaid aerodigestive tract, said at least one agent being selected fromthe group consisting of a cyanide chelator and an iron chelator.

According to another aspect of the present invention there is providedan article of manufacturing comprising a tobacco packaging material atleast a portion of which being in contact with an aerodigestive tract ofa subject in use thereof, wherein said at least a portion comprises anagent capable of reducing or preventing tobacco associated loss ofperoxidase activity in said aerodigestive tract.

According to further features in preferred embodiments of the inventiondescribed below, said tobacco packaging material comprises a filter andsaid at least one agent is impregnated in a paper of said filter beingin contact with said aerodigestive tract of said subject in use thereof.

According to still further features in the described preferredembodiments, said tobacco is smokeless tobacco.

According to still further features in the described preferredembodiments said tobacco is smoked tobacco.

According to still further features in the described preferredembodiments said tobacco packaging material is selected from the groupconsisting of a rolling paper, a filter paper, a suns bag packaging, acigarette, a pipe and a tin sheet packaging.

According to still further features in the described preferredembodiments the article of manufacturing is selected from the groupconsisting of a snuff, a cigarette, a snus, a Gutka, a plug, a twist, ascrap and tobacco water.

According to still further features in the described preferredembodiments said agent comprises a cyanide chelator.

According to still further features in the described preferredembodiments said agent is hydroxocobalamin.

According to still further features in the described preferredembodiments said agent is capable of reducing or preventing tobaccosmoke-associated death of cells in the digestive tract of a subject.

According to still further features in the described preferredembodiments said cells are lymphocytes and/or epithelial cells.

According to still further features in the described preferredembodiments said agent comprises an iron chelator.

According to still further features in the described preferredembodiments said agent is deferoxamine.

According to still further features in the described preferredembodiments said agent comprises an antioxidant.

According to still further features in the described preferredembodiments said agent is glutathione.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of preventing orreducing tobacco smoke-associated injury in the aerodigestive tract.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is schematic diagram depicting the construction of a filter paperimpregnated with agents of the present invention according to thepresent invention.

FIG. 2 a is a diagram depicting the pathway of cyanate metabolismresulting from exposure to TS-derived HCN.

FIG. 2 b is a data plot depicting reduced OPO activity following in vivoexposure (smoking) to TS in smoker and non-smoker subjects. The OPOactivity of 17 smokers and 16 non-smokers was measured prior to andfollowing smoking 1 cigarette.

FIG. 2 c is a data plot depicting reduced OPO activity following invitro exposure of saliva to TS in smoker and non-smoker subjects. TheOPO activity of 7 smokers and 11 non-smokers was measured prior to andfollowing exposure to 1 cigarette.

FIG. 3 is a histogram depicting KCSN-mediated resistance to TSexposure-induced decreases in OPO activity in saliva of non-smokersubjects exposed to the TS from 1 cigarette in vitro. Each valuerepresents the average value obtained in experiments on 3subjects±standard deviation (SD).

FIGS. 4 a-b are photographs of Western immunoblotting analysis depictingincreased levels of salivary protein carbonylation in representativenon-smoker saliva following in vivo exposure of saliva (smoking) to theTS of 1 cigarette (FIG. 4 a). Proof that equal quantities of proteinswere analyzed per sample is shown via Coomassie Blue staining (FIG. 4 h)of the samples. Abbreviations: amylase (Amy), protein rich proteins(PRP's), lysozyme (Lys). Lane 1: prior to smoking, Lane 2: 10 mins aftersmoking, Lane 3: 30 mins after smoking, Lane 4: 60 mins after smoking.

FIGS. 5 a-b are photographs of Western immunoblotting analysis depictingincreased levels of salivary protein carbonylation in representativenon-smoker saliva following in vitro exposure of saliva to the TS of 1cigarette (FIG. 5 a). Proof that equal quantities of proteins wereanalyzed per sample is shown via Coomassie Blue staining (FIG. 5 b) ofthe samples. Lane 1: prior to exposure to TS, Lane 2: 10 mins followingexposure to TS, Lane 3: 30 mins following exposure to TS, Lane 4: 60mins following exposure to TS.

FIG. 6 is a data plot depicting TS dose-dependent decrease in OPOactivity in saliva exposed in vitro to 3 exposures of TS over a 1 hperiod. Each data point represents mean±standard error of the means(SEM) of results from experiments on saliva from 4 subjects.

FIG. 7 is a data plot depicting the effect of increasing concentrationsof dapsone (4-aminophenylsulfone) on OPO activity in TS-treated saliva.Abbreviations: NO.1, NO.2, NO.3, NO4 and NO.5 correspond to saliva from5 different subjects and plasma corresponds to commercial MPO.

FIG. 8 is a data plot depicting the effect on OPO activity in salivatreated with TS in the presence or absence of 150 μM dapsone. Salivasamples were normalized with respect to initial OPO activity. OPOexposed to TS in the presence (□) or absence (♦) of dapsone, controlsaliva exposed to air in the presence (▴) or absence () of dapsone.

FIG. 9 is a data plot depicting the effect of GSH, deferoxamine (DES)and ascorbate on OPO activity in TS-treated saliva. Treatments: TS only(⋄, n=4), TS+1 mM GSH (▪, n=3), TS+1 mM deferoxamine (Δ, n=3), TS+1 mMascorbate (X, n=3). Data plotted as mean±average OPO activity.

FIG. 10 is a data plot depicting the effect of 100 μM FeCl₃ or ascorbateon OPO activity in TS-treated saliva. Data points represent data plottedas mean±SD using saliva samples obtained from 3 subjects.

FIG. 11 is a data plot depicting the effect of purified aldehydespresent in TS on OPO activity in saliva from 4-5 subjects. Treatment:exposure to air for 3 h (⋄), 2 mM acetylaldehyde (▪), 20 μMcrotonaldehyde A), 80 μM acrolein (X).

FIG. 12 is a histogram depicting the effect of 18 h of dialysis on OPOactivity in TS-treated saliva having lost 68% of initial OPO activitylevels. 1: control OPO activity in non-TS-treated saliva prior to [1]and following [2] dialysis, OPO activity in TS-treated saliva prior to[3] or following [4] dialysis and time-control following 18 h withoutdialysis [5].

FIG. 13 is a histogram depicting recovery of OPO activity in salivatreated with KCN following 18 the of dialysis. OPO activity innon-TS-treated saliva prior to [1] or following [2] dialysis, OPOactivity in saliva following 2 min KCN treatment prior to [3] orfollowing [4] dialysis and time-control OPO activity in KCN-treatedsaliva following 18 h without dialysis.

FIG. 14 is a histogram depicting OH—CO mediated prevention of OPOactivity loss in saliva resulting from TS-treatment. OPO activity innon-TS-treated saliva in the absence [1] or presence [2] of 1 mM OH—CO(n=4), OPO activity in TS-treated saliva in the absence (n=4) [3] orpresence of 0.5 mM OH—CO (n=3) [4], 1 mM OH—CO (n=3) [5] or 2 mM OH—CO(n=3) [6]. Each value represents data calculated as average±SD ofresults from 3-4 experiments using saliva from 3-4 subjects.

FIG. 15 is a histogram depicting OH—CO mediated prevention of OPOactivity loss in KCN-treated saliva. Each value represents datacalculated as average±SD of results from 3 experiments using saliva from3 subjects.

FIG. 16 is a data plot depicting death of lymphocytes incubated at 37°C. in the presence of TS and saliva.

FIG. 17 is a photograph of Western immunoblotting analysis depictingincreased levels of protein carbonylation in lymphocytes treated with TSin the presence of saliva. Lane 1: incubation in PBS alone, Lane 2:incubation in the presence of saliva, Lane 3: incubation in PBS+TS, Lane4: incubation with TS+saliva. Incubations were performed for 80 min at37° C.

FIG. 18 is a photograph of Western immunoblotting analysis depicting theeffects of saliva and uric acid on lymphocyte protein carbonylation.Lane 1: incubation in PBS alone, Lane 2: incubation in the presence ofsaliva, Lane 3:

incubation in the presence of 10 μM uric acid, Lane 4: incubation in thepresence of 100 μM uric acid. Incubations were performed for 20 min at37° C.

FIG. 19 is a data plot depicting the effects of various antioxidants; 1mM GSH, 1 mM NAC(N-acetylcysteine) and 1 mM deferoxamine (DES), onsurvival of lymphocytes incubated in the presence of TS and saliva at37° C. Asc: ascorbate.

FIG. 20 is a photograph of Western immunoblotting analysis depicting theeffects of several antioxidants on protein carbonylation levels inlymphocyte treated with TS and saliva. Lane 1: incubation in thepresence of TS and saliva, Lane 2: incubation in the presence of TS,saliva and 1 mM NAC, Lane 3: incubation in the presence of TS, salivaand 1 mM ascorbate, Lane 4: incubation in the presence of TS, saliva and1 mM GSH, Lane 5: incubation in the presence of TS, saliva and 1 mMdeferoxamine (DES). Incubations were performed at 37° C. for 20 min.

FIG. 21 is a photograph of Western immunoblotting analysis depicting theeffects of several volatile aldehydes on lymphocyte carbonylationlevels. Lane 1: incubation in the presence of PBS alone, Lane 2:incubation with 80 μM acrolein, Lane 3: incubation with 20 μMcrotonaldehyde, Lane 4: incubation with 2 μM acetaldehyde, Lane 5:incubation with 80 μM acrolein+1 mM GSH, Lane 6: incubation with 20 μMcrotonaldehyde+1 mM GSH, Lane 7: incubation with 2 μM crotonaldehyde+1mM GSH. Incubations were performed at 37° C. for 20 min.

FIG. 22 is a photograph of Western immunoblotting analysis depicting theeffects of saliva and acrolein on the lymphocyte protein carbonylationlevels. Lane 1: incubation in PBS alone, Lane 2: incubation in thepresence of saliva, Lane 3: incubation with 80 μM acrolein, Lane 4:incubation with 80 μM acrolein+saliva. Incubations were performed at 37°C. for 20 min.

FIG. 23 is a histogram depicting survival of lymphocytes incubated at37° C. for 20 min in the presence of saliva, various antioxidants andredox-active iron. Column 1: incubation in PBS alone, Column 2:incubation in the presence of saliva, Column 3: incubation in thepresence of 1 mM ascorbate, Column 4: incubation in the presence of 1 mMdeferoxamine (DES), Column 5: incubation in the presence of 90 μM FeCl₃,Column 6: incubation in the presence of saliva+1 mM ascorbate, Column 7:incubation in the presence of saliva+1 mM ascorbate+1 mM deferoxamine(DES), Column 8: incubation in the presence of 90 μM FeCl₃+1 mMascorbate, Column 9: incubation in the presence of 90 μM FeCl₃+1 mMascorbate+1 mM deferoxamine.

FIG. 24 is a histogram depicting survival of lymphocytes incubated at37° C. for 80 min in the presence of TS, ascorbate and deferoxamine(DES). Column 1: incubation in PBS alone, Column 2: incubation in thepresence of TS, Column 3: incubation in the presence of 1 mM ascorbate,Column 4: incubation in the presence of 1 mM deferoxamine, Column 5:incubation in the presence of TS+1 mM ascorbate, Column 6: incubation inthe presence of TS+1 mM ascorbate+1 mM deferoxamine.

FIG. 25 is a photograph of Western immunoblotting analysis depicting theeffects of GSH and deferoxamine (DES) on protein carbonylation levels inlymphocytes incubated for 20 or 80 minutes at 37° C. in the presence ofTS and saliva. Lane 1: incubation in the presence of TS+saliva for 20minutes, Lane 2: incubation in the presence of TS+saliva for 80 minutes,Lane 3: incubation in the presence of TS+saliva+1 mM GSH for 20 minutes,Lane 4: incubation in the presence of TS+saliva+1 mM GSH for 80 minutes,Lane 5: incubation in the presence of TS saliva 1 mM GSH+1 mMdeferoxamine for 20 minutes, Lane 6: incubation in the presence ofTS+saliva+1 mM GSH+1 mM deferoxamine for 80 minutes, Lane 7: incubationin the presence of TS+saliva+1 mM GSH+5 mM deferoxamine for 20 minutes,Lane 8: incubation in the presence of TS+saliva+1 mM GSH+5 mMdeferoxamine for 80 minutes.

FIG. 26 is a diagram depicting mechanisms of cyanate metabolismfollowing exposure to TS.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods, pharmaceutical compositions, oralcompositions, filters and tobacco products for preventing or reducingtobacco smoke-associated injury in the aerodigestive tract of a subject.Specifically, the present invention can be used to prevent or reduceloss of OPO activity or CN⁻—, redox-active metal ion- oraldehyde-induced cell death resulting from TS-associated oxidativestress, all of which being capacities not provided by prior art methods.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying examples.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or exemplified in the Examples. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Tobacco consumption, such as in the form of smoking, chewing, dipping orsnuffing, is associated with pathogenesis of many diseases of theaerodigestive tract.

Thus, various prior art methods of reducing or preventing aerodigestivetract oxidant injury resulting from insults such as TS have beendescribed.

The present inventors have previously described novel smoking filtersand oral compositions for reducing tobacco associated damage in theaeurodigestive tract (see U.S. Pat. No. 6,789,546). These compositionsinclude active agents which are capable of reducing or preventingtobacco associated loss of peroxidase activity in the aerodigestivetract.

None of these tobacco compositions and tobacco packaging means, however,are designed for reducing the damage associated with smokeless tobaccowhen in use by the subject and in particular chewing, snuff and snus.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, compositions and means for preventing or reducingany tobacco associated oxidant injury in the aerodigestive tract devoidof the above limitation.

While reducing the present invention to practice, the present inventorsuncovered that previously described agents (see U.S. Pat. No. 6,789,546)can be implemented in any tobacco and tobacco related products whichcome in direct contact with the aerodigestive tract (e.g., saliva) andas such can be ameliorate tobacco damage simultaneously with its use(e.g., chewing).

Thus, according to one aspect of the present invention there is providedan article of manufacturing comprising tobacco and a tobacco packagingmaterial, at least a portion of the tobacco and/or tobacco packagingmaterial being in contact with an aerodigestive tract of a subject inuse thereof, and whereas the at least a portion of the tobacco and/ortobacco packaging material comprises an agent capable of reducing orpreventing tobacco associated loss of peroxidase activity in theaerodigestive tract.

According to another aspect of the present invention there is providedan article of manufacturing comprising a tobacco packaging material atleast a portion of which being in contact with an aerodigestive tract ofa subject in use thereof, wherein the at least a portion comprises anagent capable of reducing or preventing tobacco associated loss ofperoxidase activity in the aerodigestive tract.

As used herein the term “tobacco” refers to any tobacco species (e.g.,crude or extract) which is compatible with human use.

On top of tobacco, the present invention also envisages the use of theagents of the present invention (in line with the above describedaspects) with other smoked, dipped, chewed, snuff or snused herbs,compatible with human consumption and which causes damage to theaerodigestive tract due to the loss of peroxidase activity.

As used herein the phrase “tobacco packaging material” refers to anyauxiliary means which packages the tobacco or facilitates itsconsumption (carrier). Examples include, but are not limited to, rollingpaper, snus bags, filter paper, tin sheets and the like.

Thus, for example, the agent may be impregnated in (attached to,absorbed in, coated with) a filter paper which comes in direct contactwith the aerodigestive tract.

FIG. 1 illustrates a cigarrete filter configuration of the tobacco smoke(TS) filter of the present invention which is referred to hereinunder asa cigarette filter 10. Cigarette filter 10 is constructed of a paperlining 12 and a filter core 14 which is composed of glass fiber and ispositioned adjacent to a tobacco filling 16. To enable effectivedelivery the agent of the present invention can be impregnated in filterlining 12. Such filters have been previously described in patentdocuments (39, 40), the teachings of which are herein incorporated byreference.

Alternatively, the rolling paper may be treated with the agent such thatthe agent is confined to that region of the paper which comes in contactwith the aerodigestive tract (say about 1 cm margines).

As used herein, “aerodigestive tract” refers to saliva-lined tissuessuch as the lips, mouth, buccal cavity, tongue, oropharynx, throat,larynx, esophagus, upper digestive tract, saliva glands, saliva, as wellas the similar mucous-lined tissues of the respiratory tract, such asthe respiratory mucosa, alveoli, trachea, and lungs.

The following provides a list of non-limiting examples of agents whichcan be used in accordance with the teachings of the present invention.

For example, as is illustrated in Examples 1 of the Examples sectionwhich follows, antioxidants such as CN⁻ chelators can be used to treatTS-associated loss of OPO activity. Examples of CN⁻ chelators, such asOH—CO and additional antioxidants which can be used by this aspect ofthe present invention are given hereinbelow.

Preferably, the CN⁻ chelator (e.g., OH—CO) is administered in a mannerwhich enables establishment of a concentration of 0.5-2 mM, preferably 1mM in body fluids, such as saliva.

CN⁻ chelators can be effectively employed to prevent or reduceTS-associated injury in the aerodigestive tract since they act tosequester CN⁻ which is injurious to OPO. Such capacity of OH—CO, alsoknown as the non-CN⁻-bound form of cyanocobalamin, hydroxocobalamin orvitamin B12a, to prevent TS-induced loss of OPO activity represents amarked improvement over prior art methods of preventing TS-mediatedoxidant injury of the aerodigestive tract since such protection hasnever been demonstrated or suggested by prior art methods employingother antioxidants such as GSH, ascorbate or deferoxamine, as shown inExample 1 of the Examples section, below.

As is illustrated in Example 2 of the Examples section which follows,antioxidants functional as redox-active metal ion chelators can be usedto treat TS-associated death of cells, such as lymphocytes.

Redox-active metal ion chelators such as deferoxamine, and physiologicalantioxidants, such as GSH are examples of antioxidants suitable for useby this aspect of the present invention, other examples are givenhereinbelow.

Redox-active metal ion chelators are used in a manner which enablesestablishment of about 1 mM concentration in body fluids (e.g., saliva).Preferably, deferoxamine is administered in a manner which enablesestablishment of a concentration of about 1 mM, more preferably about 5mM in body fluids. More preferably, a mixture of deferoxamine and GSH isused in a ratio of about 1:1, preferably 5:1, respectively. When used incombination, deferoxamine and GSH body fluid concentrations of about 1mM each are desirable although a deferoxamine concentration of 5 mM anda GSH concentration of 1 mM are also therapeutically effective. The useof antioxidants, such as GSH and redox-active metal chelators, such asdeferoxamine, preferably in combination, represents a significantimprovement over prior art methods of preventing or reducing TS-mediatedoxidant injury in the aerodigestive tract.

Examples of CN⁻ chelators suitable for use according to the presentinvention include, for example, epselen, vitamins A, C and E, seleniumcompounds, OH—CO, flavenoids, quinones (e.g., Q10, Q9), retinoids andcarotenoids.

Examples of redox-active metal ion chelators suitable for use accordingto the present invention include, for example, epselen, desferioxamine,zinc-desferioxamine, polyamine chelating agents, ethylenediamine,diethylenetriamine, triethylenetetramine, triethylenediamine,tetraethylenepentamine, aminoethylethanolamine, amino ethylpiperazine,pentaethylenehexamine, triethylenetetramine-hydrochloride,tetraethylenepentamine-hydrochloride,pentaethylenehexamine-hydrochloride, tetraethylpentamine, captopril,penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine, N,N,Bis (2aminoethyl) 1,3 propane diamine, 1,7-dioxa-4,10-diazacyclododecane,1,4,8,11-tetraaza cyclotetradecane-5,7-dione, 1,4,7-triazacyclononanetrihydrochloride, 1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and 1,4,7,10-tetraaza cyclododecane.

The iron chelator deferoxamine is also known as DES, desferal anddesferioxamine.

Articles of the present invention can further comprise at least oneflavorant such as wintergreen oil, oregano oil, bay leaf oil, peppermintoil, spearmint oil, clove oil, sage oil, sassafras oil, lemon oil,orange oil, anise oil, benzaldehyde, bitter almond oil, camphor, cedarleaf oil, marjoram oil, citronella oil, lavendar oil, mustard oil, pineoil, pine needle oil, rosemary oil, thyme oil, and cinnamon leaf oil.

The active ingredients may be introduced to the article of manufacturingas described above (e.g., snuff), such as in the form a dry powder,either as a mixture of antioxidants, or as a complex in protectiveliposomes, nanospheres or other acceptable delivery vehicles. Thispowder may be added in the final process of manufacturing and may alsocontain suitable flavors or fragrances as not infrequently used in thisindustry.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Example 1 Hydroxocobalamin Provides Protection from TobaccoSmoke-Induced Loss of Aerodigestive Tract Antioxidant Defenses

Many common and highly debilitating oral diseases, such as cancer,periodontitis and gingivitis, result from, or are aggravated byconsumption of tobacco products, such as tobacco smoking. For example,oral cancer, a frequently lethal and highly debilitating disease,results from tobacco consumption in 50-90% of cases world-wide. Onewidely accepted mechanism whereby cancer progression is promoted is viaoxidant injury, such as protein damage caused by exposure to freeradicals.

The present inventors have therefore analyzed the effects of TS onaerodigestive tract antioxidant defenses and have invented means toprevent such effects, as described below.

Exposure to Tobacco Smoke Leads to a Decrease in Oral Peroxidase and toan Increase in Protein Damage:

Oral peroxidase is the critical salivary enzymatic defense against upperdigestive tract oxidant injury resulting in, for example, macromoleculardamage associated with progression of diseases such as malignancies ofthe upper digestive tract, periodontitis and gingivitis. The mechanismof OPO mediated protection from antioxidant injury resulting fromexposure to TS HCN is schematized in FIG. 2 a.

The present inventors, as described below, have therefore analyzed theeffects of TS on OPO activity and on salivary protein carbonylation, awell-known indicator of protein damage induced by exposure to oxidativestress and/or TS (11, 12, 42).

Methods and Materials:

Saliva collection: Whole saliva was collected under non-stimulatoryconditions from healthy smokers having smoked at least 20 cigarettes aday for at least 10 years and from non-smoking subjects, as previouslydescribed (43).

Generation of tobacco smoke: Tobacco smoke was obtained from popularcommercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine(‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

In vivo exposure of saliva to TS: The OPO activity in 2 ml salivasamples of smoker and non-smoker subjects was measured prior to andfollowing the smoking of 1 cigarette. Subjects were prevented from beingexposed to TS for 1 h prior to the experiment.

In vitro exposure of saliva to TS: The in vitro exposure of salivasamples to TS was performed using a cigarette combined with a vacuumsystem, as previously described (11, 12, 14, 15). Briefly, 4-5 ml salivasamples were placed in 50 ml vaccuum flasks with a sidearm to whichcigarettes were connected. A vacuum was applied to the flasks and thesmoke from 1 cigarette was drawn into the flask in 4-5 “puffs”. Salivasamples for analysis of OPO activity were drawn immediately prior toexposure to TS and at 0, 30 and 60 min after completion of exposure ofsamples to the TS (˜10 min). Flasks were incubated in a metabolic shakerat 37° C. Immediately following collection, samples were centrifuged at800×g for 10 minutes at 4° C. to remove squamous cells and cellulardebris. The resulting supernatants were subsequently analyzed for OPOactivity and salivary protein carbonylation.

Analysis of OPO activity: OPO activity was measured according to the2-nitrobenzoic-thiocyanate (NBS-SCN) assay, as previously described(20). Briefly, DTNB is reduced to NBS by addition of β-mercaptoethanoland decreases in NBS concentration by reaction with OSCN⁻, a product ofOPO, are monitored spectrophotometrically by measuring absorbance at 412nm at pH 5.6 (18). One unit of enzyme activity was defined as theactivity required to cleave 1 μmol of NBS/min at 22° C., using a molarextinction coefficient of 12,800 (20).

Western immunoblotting analysis of protein carbonylation: Salivaryproteins were separated by SDS-PAGE using a 10% gel and electro-blottedonto nitrocellulose membranes, as previously described (14). The OxyblotKit (Intergen Co, Purchase N.Y.) was used withanti-dinitrophenyhydrazine (DNPH) antibodies to label carbonylatedproteins. Protein carbonylation levels were then visualized by reactinglabelled blots with a secondary HRP-conjugated anti-rabbit antibodyfollowed by ECL detection of secondarily labelled proteins, aspreviously described (14).

Statistical analysis of results: Ranges, means and SDs and SEMs werecomputed from the results derived from the smoker and non-smokerexperimental subgroups for the in vivo and in vitro studies. Statisticalanalysis to compare results from subgroups was performed via 2-sampleT-test for differences in means using p<0.05 as criteria to establishstatistically significant differences.

Results:

Smoking tobacco smoke leads to impaired oral peroxidase activity invivo: Following at least 1 h without exposure to TS, OPO activity levelsin saliva samples from smoker subjects were found to be 82% and 85% thatof non-smoker subjects in the subjects employed for the in vivo and invitro studies, respectively, however statistical analysis indicated thatthese differences were not statistically significant, therefore it wasconcluded that baseline OPO activity levels were similar in both smokersand non-smokers (Table 1).

TABLE 1 Basal oral peroxidase activity levels in non-smoker and smokersubjects employed for the in vitro and in vivo studies are similar. Invivo studies (U/ml) In vitro studies (U/ml) Smokers Non-smokers SmokersNon-smokers N 17 16 7 11 Range 180-1,728 193-1,019 397-1,094 236-1,166Mean 696 573 595 517 SD 417 252 237 299 SEM 101 62.9 90 91

Immediately following the smoking of 1 cigarette, a sharp drop in OPOactivity levels was observed in both smoker and non-smoker subjects,with levels dropping further in the non-smokers (42.5% of pre-smokinglevels, p<0.01) than in the smokers (58.5% of pre-smoking levels,p<0.01) (FIG. 2 b). In the absence of subsequent exposure to TS, OPOactivity levels returned to 90-100% of initial pre-smoking levels inboth groups at 30 min post-smoking.

In vitro exposure to tobacco smoke results in decreased oral peroxidaseactivity: A different set of non-smoker and smoker subjects than thoseemployed for the in vivo study were employed for the in vitro study.Similarly to the results obtained in the in vivo experiments describedabove, OPO activity levels were found to be significantly decreased atTime 0 following exposure to the TS from 1 cigarette in both studygroups with loss of OPO activity being more pronounced in non-smokersaliva than in smoker saliva (FIG. 2 c).

The literature reports that the saliva of non-smokers contains 0.3-1.5mM SCN⁻, while that of heavy smokers contains 1.4-4.0 mM SCN⁻, it waspostulated that the higher quantities of SCN⁻ in heavy smokers provideprotection against TS-induced reduction in OPO activity. Thus, in orderto ascertain why OPO activity was slightly higher and more resistant toTS in smokers relative to non-smokers, the effect of the exogenousaddition of SCN in the form of potassium thiocyanate (KSCN) tonon-smoker saliva exposed to TS was analyzed. Addition of 0.5-5.0 mMKSCN to the saliva of 3 non-smoking subjects in the in vitro system andmeasuring OPO activity before and after smoking 1 cigarette demonstratedthat addition of SCN⁻ indeed provides significant protection againstloss of OPO activity (FIG. 3).

In vivo exposure of saliva to TS causes significant salivary proteincarbonylation: In vivo exposure of saliva to TS, via smoking of 1cigarette, was found to cause significant increases in levels ofsalivary protein carbonylation, as assessed by Western immunoblottinganalysis (FIG. 4 a). The highest levels of protein carbonylation wereobserved at 10 min post-smoking (FIG. 4 a, Lane 2). The major salivaryproteins, such as amylase, acidic proline rich proteins and lysozymewere the ones found to be most carbonylated by TS.

In vitro exposure of saliva to TS causes significant salivary proteincarbonylation: Similarly to the in vivo studies described above, invitro exposure of saliva to the TS of 1 cigarette was found to causesignificant increases in levels of carbonylation of major salivaryprotein, as assessed by Western immunoblotting analysis (FIG. 5 a). Norecovery in the level of protein carbonylation was found in the in vitrostudies, as provision of new saliva is not possible.

These results therefore identify loss of OPO activity and concomitantmacromolecular damage, such as salivary protein carbonylation, asresulting from exposure to TS.

Hydrocyanic Acid (HCN) Mediates Tobacco Smoke-Associated Loss of OralPeroxidase Activity:

In order to elucidate the mechanism(s) whereby TS causes loss of OPOactivity, the effect of various oxidants and antioxidants on OPOactivity in saliva exposed to TS in vitro were analyzed, as describedbelow.

Materials and Methods:

Generation of tobacco smoke: Tobacco smoke was obtained from popularcommercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine(‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

In vitro exposure of saliva to TS: The in vitro exposure of salivasamples to TS was performed as described above with the modificationthat the same saliva samples were subjected to multiple exposures to theTS of 1 cigarette at 20 min intervals.

Analysis of OPO activity: Analysis of OPO activity in TS-treated salivasamples was performed as described above.

Treatment of saliva with TS in the presence of oxidants, antioxidants,and inhibitors: The various reagents and materials were added to salivaprior to treatment with TS at the specified concentrations. Unlessotherwise specified, all chemicals were obtained from Sigma ChemicalCorp. (St. Louis, Mo., USA).

Statistical analysis: Compilation and statistical analysis of resultswas performed as described above for in vitro studies.

Results:

Exposure of saliva to the TS of 3 cigarettes resulted in a TSdose-dependent decrease in OPO activity to 22% of pre-TS exposure levels(FIG. 6).

Dapsone a, has been shown to specifically inhibit SPO but not MPO atacidic pH (44). Thus, in order to determine which of the MPO or SPOactivity components of OPO activity are lost as a result of exposure toTS, OPO activity in TS-treated saliva was measured in the presence ofincreasing concentrations of 50-150 dapsone. Loss of OPO activity due toexposure to TS was found to be 60-85% in the presence of 150 μM ofdapsone whereas control MPO activity was unaffected, (FIG. 7) and lossof OPO activity was similar in the presence or absence of 150 μM dapsonein saliva containing ˜40% MPO (FIG. 8) indicating that TS affects SPOand MPO activities to a similar extent.

In the presence or absence of 1 mM of ascorbate or the antioxidants GSHor deferoxamine, loss of OPO activity in TS-treated saliva was found tobe similar (FIG. 9) and exposure of saliva with 1 mM ascorbate or theoxidant 100 μM FeCl₃ for 2 h did not inhibit OPO activity (FIG. 10).

In the presence of 2 mM 80 μM acrolein, and 20 μM crotonaldehyde, majoraldehydes present in TS, OPO activity in TS-treated saliva wasunaffected (FIG. 11).

Levels of OPO activity in TS-treated saliva having lost ˜68% of initialOPO activity were restored to 94% of initial levels following subsequenttreatment with 18 h of dialysis (FIG. 12). Thus, loss of OPO activity inTS-treated saliva can be treated, presumably, via removal of lowmolecular weight molecules.

Cyanide is a known inhibitor of heme peroxidase, and the gas-phase TS ofvarious cigarette brands have been found to contain 2-233 μg of KCN(45). Thus, in order to test the hypothesis that dialysis treatmentrestores OPO activity in TS-treated saliva via removal of KCN, salivasamples were treated with 150 μM KCN and analyzed for OPO activity. Thisconcentration of KCN was observed to cause a loss of ˜65% of OPOactivity after only 2 min of incubation, which loss was considerablyreversed by dialysis (FIG. 13), thereby indicating that KCN is indeedcapable of inhibiting OPO activity.

Hydroxocobalamin prevents tobacco smoke-associated loss of salivaryperoxidase: Since CN⁻ ion was found to be involved in TS-associated lossof the OPO activity, the present inventors have attempted to provide ameans of preventing such loss of OPO activity via the use of a CN⁻chelator.

Addition of increasing amounts of OH—CO, a known chelator of CN, wasshown to prevent, to a significant extent, the loss of OPO activitysaliva 40 min after treatment with TS (FIG. 14). Similarly, addition of1 mM OH—CO to saliva treated for 2 min with 1 mM KCN was sufficient tocompletely prevent the KCN-associated loss of 40-60% of OPO activity(FIG. 15). Furthermore, preincubation of saliva with OH—CO could preventboth TS- and KCN-associated loss of OPO activity, indicating thatcyanide indeed mediates TS-associated loss of OPO activity.

These results therefore indicate that, according to the presentinvention, OH—CO can be employed to effectively reduce or prevent theoccurrence of diseases, such as malignancies of the aerodigestive tract,periodontitis and gingivitis associated with exposure to TS.

Example 2 The Antioxidants Deferoxamine and Glutathione Prevent UpperAerodigestive Tract Lymphocyte Death Associated with Exposure to TobaccoSmoke

Many diseases of the aerodigestive tract are associated with consumptionof tobacco products or betel nut chewing. For example, oral SCC is themost common malignancy of the head and neck, having a high rate ofmorbidity and mortality. In as many as 90% of the cases, this cancer isinduced by exposure of oral epithelial tells to tobacco products such asTS, or chewed betel nut. This exposure always occurs in the presence ofsaliva and presumably is induced by free radicals. Thus, the effects ofTS on cells in the presence of saliva was examined, as described below.

Materials and Methods:

Mutagenic alterations of oral mucosal cells induced by TS occur in thepresence of saliva (22, 23). Thus, in order to explore the interactionbetween TS and saliva on cells, intact cells were exposed to TS, aloneor in the presence of saliva. Lymphocytes were employed since they arehighly sensitive to oxidant injury.

Saliva collection: Saliva was collected from healthy subjects, 3 malesand 3 females ranging from 21-47 years of age, under non-stimulatoryconditions in a quiet room during the morning between 8 am and noon.Saliva collection was performed at least 1 h after eating by spittinginto a recipient for 10 minutes, as previously described (43). Followingcollection, saliva was immediately centrifuged at 800×g for 10 min at 4°C. to remove cells and cell debris and the resulting supernatant wasused for biochemical analyses.

Lymphocyte isolation: Blood from 10 consenting, healthy, non-smokingsubjects, 5 males and 5 females ranging from 18-55 years of age, wasdrawn into EDTA-containing vacutainers. Lymphocytes were isolated byFicoll-Hypaque (Sigma) gradient centrifugation according to themanufacturer's instructions and lymphocytes were suspended at 10⁷cells/ml PBS (Beit-Ha'emek Industries, Israel), and used immediately inexperiments.

Generation of tobacco smoke: Tobacco smoke was obtained from popularcommercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine(‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

Treatment of lymphocytes with TS in the presence of saliva: Exposure oflymphocytes to TS was carried out by attaching a Time cigarette (capableof removing particles>0.1 mm in diameter) in which the filter tip wasremoved to a Cambridge filter which was combined with a vacuum system toaspirate gas-phase TS inside sealed 250 ml flasks containing lymphocytessuspended in 12-15 ml PBS, as previously described (14, 15). Areproducible vacuum was created in the flask and upon application ofvacuum for 5 s, 80-100 ml of TS from the lit cigarette was drawn intothe flask. The chemical addititives GSH, NAC, deferoxamine, ascorbate(Asc), uric acid or FeCl₃ (Sigma) were added at the specifiedconcentrations. Saliva treatment of lymphocytes was performed bysuspending these in PBS supplemented with 30% (v/v) saliva. Followingtreatment with the TS of 0.5 cigarettes, flasks were incubated at 37° C.for 20 min in a metabolic shaker and subjected to a further 4 treatmentswith TS, as described above.

Measurement of lymphocyte survival: Lymphocyte viability was assessed byTrypan Blue exclusion assay.

Western immunoblotting analysis of lymphocyte protein carbonylation:Lymphocytes were washed twice in PBS following treatment bycentrifugation at 2,000 rpm for 2 minutes to remove saliva and othercomponents of the incubation medium. Lymphocytes were then centrifugedat 14,000 rpm for 1 minute and lysed by sonication for 10 seconds inlysis buffer containing 20 mM Tris pH 7.4, 1 mM EGTA, 1 mM PMSF, 50 μMNaVO₄ and 50 mM NaF. Lysates were centrifuged at 14,000 rpm for 1 minuteand supernatants were harvested for analysis. Carbonylation analysis wasperformed as described above.

Statistical analysis: Results for statistical analysis were obtainedfrom the control subgroup (lymphocytes in PBS) and from the varioustreatment subgroups. Means, SDs and SEMs were computed and resultsbetween the subgroups were analyzed and compared via one-wayanalysis-of-variance (46) using the Bonferroni Multiple-Comparison TestModel (47) to determine significant differences between computed means.The means between each pair of means was analyzed via T-test For PairedDifferences and means between each two subgroups were compared via TwoSample T-test For Differences in Means (48).

Results:

During an 80 minute incubation of lymphocytes in PBS alone or in PBSsupplemented with 30% (v/v) saliva, neither cell death nor proteincarbonylation (FIGS. 16 and 17, respectively) occurred. Treatment oflymphocytes with TS in the absence of saliva resulted in time-dependentincreases in cell death and protein carbonylation levels, peaking after80 minutes with a death rate of 44% (p<0.01). Addition of saliva tolymphocyte suspensions during TS treatment synergistically potentiatedthe lethal effect of the TS, as demonstrated by an 86.2% (p<0.01) deathrate and highly elevated protein carbonylation levels (FIGS. 16 and 17,respectively).

Addition of saliva to lymphocytes incubated for 80 minutes in PBS aloneresulted in a mild reduction of protein carbonylation levels (FIG. 17).This was shown to be due to uric acid, the major non-enzymaticantioxidant in saliva (18, 28, 49), since addition of uric acid to thesuspension medium instead of saliva led to reduced carbonylation levels(FIG. 18).

Supplementation of suspension medium with 1 mM ascorbate or 1 mM NAC didnot alter the death rate of lymphocytes exposed to TS and saliva,whereas, addition of 1 mM GSH partially prevented this lymphocyte loss.At 20 and 80 minutes following TS and saliva treatment, the lymphocytesurvival rates dropped to 57.1% and 13.8% respectively, whereas in thepresence of GSH these survival rates were 93.4% (p=0.0001) and 24.3%(p=0.0037) respectively (FIG. 19).

Similarly to GSH treatment, addition of 1 mM deferoxamine significantlyprevented lymphocyte death at 80 minutes, with a survival rate of 23.7%(p=0.005) (FIG. 19).

The unique protection offered by GSH at 20 minutes, in contrast to thelack of protection by all other antioxidants examined, was alsodemonstrated by assessing lymphocyte protein carbonylation levels (FIG.20).

Role of aldehydes: The demonstrated protection from TS+saliva-inducedlymphocyte death by GSH led to the hypothesis that this effect wasmediated by aldehydes associated with TS. Indeed, addition of variousexogenous aldehydes known to be present in TS to lymphocytes suspendedin PBS similarly led to cell death. Namely, in the presence of 80 μMacrolein, 20 μM crotonaldehyde or 2 mM acetaldehyde survival rates after20 minutes were 63.3%, 80.3% and 93.6%, respectively, and at 80 minutesthese death rates were 57.3%, 75.3% and 92.7%, respectively. Thismimicking of the TS+saliva-induced lethality by exogenous aldehydes wasfurther supported by protection from cell death being mediated byaddition of GSH, similarly to GSH-mediated viability restoration in thepresence of TS and saliva. Addition of GSH in the presence thesealdehydes resulted in lymphocyte survival rates at 20 minutes of 93.3%,82.3% and 93.3%, respectively and at 80 minutes these survival rateswere 87.4%, 81.3% and 93.6%, respectively. As the survival rate ofcontrol lymphocytes was 95.7%, it was concluded that acetaldehyde doesnot play a role while acrolein, and to a slightly lesser degreecrotonaldehyde, are the aldehydes causing cell death.

The pattern of injury induced by these aldehydes and the protectionprovided by GSH was similar in the concomitant levels of proteincarbonylation (FIG. 21). However, in contrast to the profoundsynergistic effect demonstrated by treatment with saliva and TS,treatment with acrolein, the most potent thiol demonstrated, with salivadid not significantly alter the survival rates (57.9% and 66.4%respectively) and did not enhance carbonylation levels (FIG. 22). Thisruled out the possibility that TS-saliva-based synergism involvesTS-based aldehydes.

Role of transition metal ions: The relatively moderate impact whichexogenous aldehydes had on lymphocyte survival and the previously notedlack of related aldehyde-saliva synergy raised the possibility thatthere might be another mechanism mediating the injurious effects of TSon lymphocyte survival. The fact that deferoxamine had a similarprotective effect as GSH pointed to the possible role of redox-activeiron ions. In order to test this hypothesis, the role of redox-activeiron was analyzed via an assay employing detection of redox-active ironmediated potentiation of ascorbate pro-oxidant activity. The role ofredox-active iron ions was also analyzed using deferoxamine, a verypotent iron chelator.

Addition of ascorbate to saliva-containing medium without exposure to TSresulted in reduction of the lymphocyte survival rate to 78% (p<0.01;FIG. 23, column 6). The modulatory role of redox-active iron was furtherdemonstrated by addition of ascorbate to PBS containing iron but notsaliva, as FeCl₃. This yielded similar results to those obtained byaddition of ascorbate to saliva. Reduction of the lymphocyte survivalrate to 77% was demonstrated (FIG. 23, Column 8), which reduction wastotally prevented in the presence of deferoxamine (FIG. 23, Column 9).

Neither ascorbate nor deferoxamine were found to have any modulatoryeffect on the direct moderate lethal effect which TS had on lymphocytesin the absence of saliva (61% survival rate) (FIG. 24). This proved thatredox-active iron ions did not originate from the TS but rather from thesaliva, as described.

Protection from TS-saliva mediated lymphocyte death by GSH anddeferoxamine: Since the two major underlying mechanisms of lymphocyteloss identified above were based on the action of aldehydes and highlyreactive free radicals mediated by redox-active iron, experiments aimedat protecting lymphocytes from both injurious mechanisms simultaneouslywere performed as follows.

Lymphocytes were exposed to TS and saliva in the presence of 1 mM GSH orboth 1 mM GSH and deferoxamine at either low (1 mM) or high (5 mM)concentration. Both survival rates and carbonylation levels wereexamined after 20 and 80 minutes. Very significant protection againstthe lethal effect of TS was obtained by addition of 1 mM GSH and 5 mMdeferoxamine to the saliva-containing medium prior to TS treatment ofthe lymphocytes. Whereas the survival rates of the non-protectedlymphocytes were 57.2% and 14.4% at 20 and 80 minutes respectively,these rates climbed to 90.8% (p=0.0001) and 61.6%, respectively (Table2). Furthermore, concomitant with this very significant improvement insurvival rate, protein carbonylation levels dropped to nearly nil (FIG.25, lanes 7,8).

TABLE 2 Effect of GSH alone or in combination with 1 mM deferoxamine on% survival of TS + saliva-treated lymphocytes. 20 minute treatment 80minute treatment Treatment Mean SEM p Mean SEM p — 57.2 3.7 14.4 2.6 1mM GSH 90.6 2.1 0.0001 28.8 3.4 0.0001 1 mM GSH + 92.2 1.6 0.0001 57.45.3 0.0001 1 mM deferoxamine 1 mM GSH + 90.8 2.7 0.0001 61.6 3.0 0.00015 mM deferoxamine

A comprehensive mechanism for the induction of disease by TS issuggested in which a new role is attributed to saliva losing itsantioxidant capacity and becoming a potent prooxidant milieu in thepresence of TS (FIG. 26). This mechanism is based on the resultsobtained in the current study as well as on the well-known observationthat oral cancer mostly occurs in oral epithelial cells exposed totobacco products (from TS or chewing betel nut) in the presence ofsaliva.

These results therefore indicate that GSH and deferoxamine, according tothe present invention, can therefore be effectively employed to preventor reduce diseases of the aerodigestive tract associated with cell deathcaused by tobacco consumption.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES CITED

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1. A article of manufacturing comprising tobacco and a tobacco packagingmaterial, at least a portion of said tobacco and/or tobacco packagingmaterial being in contact with an aerodigestive tract of a subject inuse thereof, and whereas said at least a portion of said tobacco and/ortobacco packaging material comprises at least one agent capable ofreducing or preventing tobacco associated loss of peroxidase activity insaid aerodigestive tract, said at least one agent being selected fromthe group consisting of a cyanide chelator and an iron chelator.
 2. Anarticle of manufacturing comprising a tobacco packaging material atleast a portion of which being in contact with an aerodigestive tract ofa subject in use thereof, wherein said at least a portion comprises anagent capable of reducing or preventing tobacco associated loss ofperoxidase activity in said aerodigestive tract.
 3. The article ofmanufacturing of claim 1, wherein said tobacco packaging materialcomprises a filter and said at least one agent is impregnated in a paperof said filter being in contact with said aerodigestive tract of saidsubject in use thereof.
 4. The article of manufacturing of claim 1,wherein said tobacco is smokeless tobacco.
 5. The article ofmanufacturing of claim 1, wherein said tobacco is smoked tobacco.
 6. Thearticle of manufacturing of claim 1, wherein said tobacco packagingmaterial is selected from the group consisting of a rolling paper, afilter paper, a suns bag packaging, a cigarette, a pipe and a tin sheetpackaging.
 7. The article of manufacturing of claim 1, selected from thegroup consisting of a snuff, a cigarette, a snus, a Gutka, a plug, atwist, a scrap and tobacco water.
 8. The article of manufacturing ofclaim 1, wherein said cyanide chelator is hydroxocobalamin.
 9. Thearticle of manufacturing of claim 1, wherein said agent is capable ofreducing or preventing tobacco smoke-associated death of cells in thedigestive tract of a subject.
 10. The article of manufacturing of claim9, wherein said cells are lymphocytes and/or epithelial cells.
 11. Thearticle of manufacturing of claim 1, wherein said iron chelator isdeferoxamine.
 12. The article of manufacturing of claim 1, wherein saidat least one agent further comprises an antioxidant.
 13. The article ofmanufacturing of claim 12, wherein said antioxidant is glutathione. 14.The article of manufacturing of claim 1, wherein said tobacco packagingmaterial comprises a filter and said at least one agent is impregnatedin a paper of said filter being in contact with said aerodigestive tractof said subject in use thereof.
 15. The article of manufacturing ofclaim 2, wherein said tobacco is smokeless tobacco.
 16. The article ofmanufacturing of claim 2, wherein said tobacco is smoked tobacco. 17.The article of manufacturing of claim 2 wherein said tobacco packagingmaterial is selected from the group consisting of a rolling paper, afilter paper, a suns bag packaging, a cigarette, a pipe and a tin sheetpackaging.
 18. The article of manufacturing of claim 2, wherein said ronchelator is deferoxamine.
 19. The article of manufacturing of claim 2,wherein said at least one agent further comprises an antioxidant. 20.The article of manufacturing of claim 19, wherein said antioxidant isglutathione.